This invention relates to the use of mass spectrometry as a means for identifying specific colonization factors 9CF) in a sample of E. coli. The method is useful for tracking infections by differentially identifying the CF produced by specific organisms.
Colonization factors (fimbriae, fibrillae or pili) are important virulence determinants of both intestinal and extra-intestinal Escherichia coli. For example, enterotoxigenic E. coli (ETEC), a major causative agent of diarrhea in children from and travelers to endemic regions, utilize CF for initial adherence necessary for colonization. However, the ability to detect and differentiate CF has been problematic, with incomplete CF data commonly being reported from field and survey studies. In addition, while efforts to clone and sequence E. coli CF have been underway for almost two decades, sequences of the same CF have been published with disagreement at several bases, indicating different amino acids at those positions. An independent means of verification of these sequences would be very valuable. This invention provides a technique of subunit mass determination that provides for the identification of E. coli CF as well as verification of sequence data.
The use of mass spectrometry for purposes of identifying colonization factors (CF) was first suggested by Cassels in an Abstract published in 1993. However, the methods suggested therein were not so effective as those under consideration in this disclosure, since the optimization achieved by following the steps taught herein provide increased efficiency and reliability.
Strains of ETEC and enteropathogenic E. coli (EPEC) were grown on agar or liquid broth, surface proteins removed, and CF detected by SDS-PAGE. Extracts containing sufficient amounts of CF were partially purified, with samples examined by electrospray mass spectrometry (MS) for mass determination of the CF subunit. In some cases N-terminal protein sequence (PSQ) of the subunit was obtained. In addition, purified CF from uropathogenic E. coli (P pili) were examined by MS. Results indicated that the use of MS gave a definitive identification of E. coli CF in almost all cases. Utilizing a simple process of growing ETEC strains, recovering CF, and purifying CF, 28 CF from 30 ETEC strains were identified. Overall eighteen different CF subunits were examined by MS. MS data for twelve CF subunits indicated agreement with published data (three validated one published sequence over another), and six of the CF are not yet sequenced.
The data clearly shows that mass spectrometry provides for identification of CF from ETEC strains grown in a simple standardized procedure. Moreover, MS data can be used to verify sequencing data of E. coli CF, as well as give the mass of unsequenced CF for future comparison. The resolving power of MS is such that the structural subunits of E. coli CF are differentiable, with protein sequencing providing valuable confirmatory information.
The preferred process for identifying bacterial colonizing factors in a culture comprises the steps of:
1) suspending bacteria in an isotonic solution, followed by heating for 15 to 30 minutes at a temperature sufficient to release the colonization factors into solution,
2) centrifuging the product obtained in step 1, then discarding the precipitate obtained after centrifugation while retaining the supernatant,
3) adding sufficient ammonium sulfate to the supernatant obtained in step 2 to obtain a concentration of 15% to 50% saturation of ammonium sulfate until a precipitate is seen,
4) centrifuging the product of step 3 containing the precipitate to pelletize the precipitate,
5) dissolving the pellet obtained in step 4 in water and dialyzing to remove ammonium sulfate and other small molecules and retaining the material remaining inside the dialysis membrane,
6) drying the product retained in the dialysis membrane in step 5 to obtain dried colonization factor,
7) solubilizing the dried colonization factor obtained in step 6 by first dissolving in 1,1,1,3,3,3-hexafluoro-2-propanol, then adding a volatile acid in aqueous solution to provide solubilized colonization factor,
8) subjecting solution containing solubilized colonization factor obtained in step 7 to mass spectrometry to determine mass, and comparing mass of proteins found therein with mass of known colonization factors.